The development strategy for an effective tetravalent dengue vaccine has consisted of the clinical evaluation of monovalent vaccine candidates for each of the four serotypes, with the goal of selecting suitable candidates for inclusion in a tetravalent formulation. Previously, monovalent vaccine candidates were evaluated in a combined total of over 500 human subjects to determine safety, infectivity, and immunogenicity. These studies identified a collection of 6 suitable vaccine candidates: DEN1del30, DEN2/4del30, DEN3del30/31, DEN3-3D4del30, DEN4del30, and DEN4del30-200,201. The selected vaccine candidates were safe and well-tolerated, elicited a robust antibody response in more than 80% of subjects, and were highly infectious (50% infection dose < 10 PFU). Tetravalent studies: Previously, several Phase I clinical studies were completed to evaluate four different tetravalent formulations containing 1000 PFU of each vaccine candidate. For each of the formulations, the frequency of seroconversion was as high as 85 to 100% for serotypes 1, 3, and 4. However, the frequency of seroconversion only reached 65% for serotype 2. During the past year, we sought to increase this level of DENV-2 seroconversion using two approaches: generate a fifth formulation (TV-005) containing a 10-fold higher dose of the DEN2/4del30 component and administer a second dose of each formulation at 6 months following the primary dose. Following a single dose of TV-005, the frequency of seroconversion to DENV-2 increased by only 10%. Surprisingly, a second dose of any of the formulations did very little to increase seroconversion and did not boost neutralizing antibody titers, indicating that the primary dose elicited sterilizing immunity capable of completely neutralizing the second dose of vaccine. Following the second dose, vaccine viremia was not detectable in any volunteer, whereas viremia was detected in 60 to 85% of subjects following the primary dose. These data suggest that a single dose of vaccine may be sufficiently immunogenic. This is substantiated by the finding that the preferred formulation (TV-003) elicits a trivalent or greater response in 90% of subjects. Clinical evaluation of TV-003 in an expanded number of both naive and flavivirus sero-positive subjects is currently underway. Should future studies of this vaccine prove it to be efficacious, the vaccine could be a cost-effective means of controlling dengue in endemic areas and a tremendous public health asset.